Protein tyrosine kinases (PTKs) play a key role in cell signaling and regulating biological processes such as proliferation, differentiation, and apoptosis. More than 164 protein kinases have been implicated in diseases including cancer, inflammation, diabetes, hypertension, angiogenesis, and immune disease. Directly or indirectly, over 400 diseases have been linked to protein kinases. In light of this, PTKs have become attractive targets for drug development in both academia and the pharmaceutical industry. Despite the large number of targets, however, the FDA-approved kinase inhibitiors as well as those in clinical development target only a handful of well-known kinases providing inhibition via competition with ATP. Clearly, obtaining potent and selective ATP-competitive compounds against a wide-variety of tyrosine kinases has been difficult. This proposal aims to develop general and robust tools for the rapid identification of substrate-competitive protein tyrosine kinase (PTK) inhibitors. All current approved therapeutics targeting inhibition of PTKs involved in disease are ATP-competitive inhibitors. However, we believe that substrate-competitive inhibition of PTKs offers several important benefits over ATP-competitive inhibition. Due to very high similiarity in the ATP binding pocket across PTKs, obtaining selective inhibition is particularly challenging. Selective inhibition of a target kinase will minimize off-target side-effects of therapeutics as well as provide selective chemical probes for biological studies. Substrate-competitive inhibitors represent an advance toward selective kinase inhibition owing to the non-homologous substrate pocket of PTKs. We plan to develop our methodology using four initial targets: c-Abl, c-Src, and two essential tyrosine kinases in Trypanosomes. c-Abl and c-Src are both involved in cancer progression (validated targets for chronic myelogenous leukemia (CML) and colon cancer, respectively) and the Trypanosome PTKs are potential therapeutic targets for African sleeping sickness (human African trypanosomiasis, HAT). CML is a rare, inoperable cancer which results in more than 5,000 new cases per year in the United States. Colon cancer is the third most common cancer, resulting in over 650,000 deaths per year. African sleeping sickness is a growing problem in western Africa, resulting in 300,000 new cases per year and over 60,000 deaths per year. We believe the proposed research is well-suited for the EUREKA program because: 1) it develops cutting- edge methodologies which challenge the widespread paradigm of kinase inhibition via ATP-competitive compounds, and 2) creates a flexible and robust platform for obtaining substrate-competitive kinase inhibitors against any kinase target involved in disease progression.